E. coli p-aminobenzoate synthetase (PABS) and anthranilate synthtase (AS) are structurally related enzymes that catalyse slightly different reactions. The experiments in this proposal are designed to determine and characterize nucleotide and amino acid sequence differences responsible for the altered functional properties of the two enzymes. AS has been extensively characterized. The aims of this proposal are to obtain pabA and pabB on recombinant plasmids. The nucleotide sequences of pabA and pabB will be determined and compared to the genes encoding AS (trpE and trp(G)D). Hybrid genes will be constructed between pabA and trp(G)D and between pabB and trpE, by methodology involving both recombinant DNA techniques and in vivo recombination. The hybrid gene products will be characterized for functional alterations both in vivo and in vitro. In vivo characterization will include selection for hybrid gene products with specific functional properties. In vitro analysis will concentrate on biochemical parameters of the hybrid gene products. The results of the proposed studies will include information on the way in which genes evolve following a duplication of genetic information. The extent and type (neutral vs. functional) of nucleotide and amino acid sequence divergence will be estimated, and the presence of functional domains on the polypeptide chains will be tested. The methodology is unique in that a functional as well as comparative approach to evolution is described, allowing a direct evaluation of the consequences of nucleotide and amino acid sequence divergence.